We next compared the effect of mutating
RRP6 and SSU72 alone or together on the genomic profile of coding and ncRNAs. Total
RNA from wild-type (WT), ssu72-2, Drrp6, and
double ssu72-2Drrp6 strains grown at 32°C
(semipermissive conditions) was hybridized to
strand-specific Saccharomyces cerevisiae tiling
arrays. The profiles obtained confirmed that loss
of Rrp6 causes accumulation of CUTs, especially from bidirectional promoters (7). However, ssu72-2 mutation alone or in combination
with Drrp6 gave rise to many additional ncRNAs
(Fig. 2A).

Ssu72 is involved in the transcription termination of small nucleolar RNAs, as is clearly
revealed by the widespread appearances of extended transcripts for these genes in ssu72-2 (fig.
S2A, I) (14). The profiles also unveil a role of
Ssu72 in transcriptional termination of CUTs, as
many show 3′ extensions in the ssu72-2Drrp6
double mutant compared with Drrp6 (fig. S2A,
II). Ssu72 inactivation also leads to increased
initiation of new cryptic transcripts. Like CUTs,
Ssu72-restricted transcripts (SRTs) often run in a
divergent orientation from bidirectional promoters. We detected some SRTs in the single ssu72-2
mutant strain and others only in combination with
RRP6 deletion (Fig. 2A). The array data demonstrated the presence of 605 SRTs in addition to
the expected 1982 CUTs (Fig. 2B), as validated
in specific cases (fig. S2B).

CUT and SRT initiation is associated withmRNA transcription start sites (TSSs) (Fig. 2B)(6, 7). To focus on promoter-associated ncRNAs(pncRNAs), we selected CUTs and SRTs that arepositioned between tandem open reading frames(ORFs) (hereafter referred to as pCUTs and pSRTs).678 pCUTs and 135 pSRTs initiate antisensetranscription between tandem ORFs. Promotersthat generate a divergent pncRNA tend to expressmore mRNA (down ORF) (Fig. 2C). In contrast,we found no correlation in mRNA expression level(up ORF) with downstream-positioned ncRNAs.We further showed that SRT expression is not dueto loss of nuclear pre-mRNA down-regulation–dependent CUT termination or differential RNAstability effects (fig. S3, A and B). Finally, a genome-wide Pol II occupancy profile of the ssu72-2 mu-tant (12) revealed a distinct peak upstream of theTSS, which is absent in the wild type (Fig. 2D),as well as a higher Pol II occupancy over SRTtranscript regions in the mutant compared withthe wild type (fig. S3C). Overall these results es-tablished that the loss of Ssu72 promotes de novoinitiation of pSRTs.